【作者】 周伟；

【导师】 杨辉；

【作者基本信息】 第三军医大学 ， 外科学， 2011， 博士

【摘要】 垂体腺瘤(Pituitary adenoma, PA)是神经内分泌系统常见的一种肿瘤,也是颅内常见的良性肿瘤(约占颅内肿瘤的10%~20%)。该肿瘤虽为良性,部分肿瘤却可突破包膜,侵犯邻近的骨质、海绵窦等周围组织,呈侵袭性生长,为侵袭性垂体腺瘤。这类侵袭性肿瘤,由于其术后残留的肿瘤组织的生长,手术将其完全切除的难度较大,且术后易复发,其病人的预后与肿瘤侵袭的程度密切相关。研究这类肿瘤侵袭性的调控对于评估患者的预后,探索新的辅助治疗方法有着重要的意义。雌激素受体(Estrogen Receptor, ER)作为雌激素发挥作用的效应分子,通过激活相关的细胞内信号分子,参与腺垂体的细胞增殖及激素分泌。雌激素受体属于类固醇核受体超家族中的成员,分为ERα及ERβ两种亚型。研究表明ERα能通过上调小鼠生长激素型垂体瘤GH3细胞系的促生长基因,促进细胞的生长。另外,缺失了ERβ的雌性小鼠生长到24月龄时,出现了促性腺激素类型的垂体腺瘤,而雄性的ERβ基因敲除小鼠、ERα敲除的小鼠及ERα、β双敲的小鼠中,都未出现这一现象。这些研究结果表明ERα与ERβ的失衡,对垂体腺瘤的发生及生物学特性有着极为重要的影响。但其机制尚需进一步的研究。大量的研究表明,相邻的细胞间连接减弱或消失,产生细胞分离现象是肿瘤的侵袭性发展至关重要的一步。细胞粘附分子上皮细胞钙粘蛋白(E-cadherin)表达的高低与多种肿瘤的侵袭性及转移能力有非常密切的关系。有研究表明在生长激素型及泌乳素型垂体腺瘤中,侵袭性较高的肿瘤胞浆的E-cadherin表达明显下调。说明E-cadherin的表达水平与垂体腺瘤的侵袭性存在密切的相关性。经证明在一些细胞系中ER的激活能够影响E-cadherin的表达,然而这种作用是否存在于垂体腺瘤中,目前缺乏相关方面的研究。E-cadherin的表达主要受其抑制子Slug的直接调控。体内及体外研究表明,在肿瘤形成过程中E-cadherin的表达与Slug呈负相关,并对病人的预后有着极大的影响。虽然在很多上皮来源的肿瘤中,Slug通过对E-cadherin的抑制以影响肿瘤侵袭性得到了证实,但在垂体腺瘤中Slug的调控作用则知之甚少。无功能性腺瘤(Nonfunctional pituitary adenoma, NFPA)是垂体腺瘤中最为常见的类型之一,与其他类型的垂体腺瘤相比,NFPA临床上内分泌症状不明显。本研究以NFPA为研究对象,检测侵袭性及非侵袭性NFPA组织中ER、Slug及E-cadherin的表达,及它们之间的关系,分析其与侵袭性之间的相关性。并通过不同的ER激动剂作用于原代培养的NFPA细胞,近一步研究ER影响侵袭性的机制。本研究包括两部分:第一部分雌激素受体在无功能性垂体腺瘤中的表达及其与肿瘤侵袭性的关系实验对象为新桥医院收入的41例NFPA患者手术标本。采用免疫组化的方法检测ERα、ERβ、增殖指标PCNA及Ki-67,并分析其与NFPA侵袭性的关系。同时从mRNA及蛋白水平检测样本中E-cadherin与Slug的表达,分析其可能参与NFPA侵袭性调控的机制。主要结果如下:1.临床资料分析数据显示,被研究的女性患者中侵袭性肿瘤的发生率为55%,男性组的发生率为16%,与男性组相比,女性组侵袭性NFPA的发生率增高。侵袭性肿瘤中女性组血清雌激素水平与男性组的水平无统计学差异。在侵袭性NFPA中,女性组的ERα表达量高于男性组。2. 41例NFPA总样本中37例为ERα阳性(90%),其中15例侵袭性肿瘤全为ERα阳性,且侵袭性肿瘤的ERα表达量明显高于非侵袭组。总样本中31例为ERβ阳性(76%),其中26例非侵袭性肿瘤中25例为ERβ阳性,15例侵袭性肿瘤中6例为阳性(60%)。与ERα相反,侵袭性肿瘤的ERβ表达量明显低于非侵袭组。3. 41例样本瘤体组织中PCNA表达量都很高,其中侵袭组PCNA标记指数与非侵袭组间无统计学差异(P=0.534)。与PCNA标记指数相似,Ki-67标记指数在两组无统计学差异(P=0.807)。相关性分析显示,两个增殖指标与ERα及ERβ均无相关性。4. mRNA水平及蛋白水平结果相一致,经比较侵袭组E-cadherin表达量较非侵袭组明显降低,Slug蛋白的表达量明显增高。相关性分析结果显示E-cadherin的表达与Slug的表达呈负相关;E-cadherin与ERα间呈负相关,与ERβ呈正相关;与之相反,其抑制子Slug与ERα间呈正相关,与ERβ呈负相关。推测ER可能通过E-cadherin-Slug通路影响NFPA的侵袭性。第二部分雌激素受体对无功能性垂体腺瘤细胞侵袭性的影响雌激素受体属于甾体激素,属于配体依赖性转录因子。ER自然的内源性配体为17β-雌二醇(E2),除此以外,一些合成的化合物如PPT、DPN还能选择性的激活ERα和ERβ。本研究选择侵袭性无功能性垂体腺瘤细胞为对象,对其进行原代培养并鉴定,应用免疫荧光双标方法检测ERα和ERβ的表达情况,给予选择性激动剂作用细胞,观察其对细胞的增殖、侵袭的影响情况并初步探讨其机制。主要结果如下:1.原代细胞培养细胞的来源为侵袭性NFPA组织,这些细胞中ERα与ERβ均有表达,与ERβ的表达相比,ERα的表达处于优势,这与第一章中组织检测结果相一致,免疫荧光双标结果显示两种受体在细胞中的分布差异明显,共表达两种受体的细胞较少见。2.利用MTT法绘制生长曲线,检测了ER的不同激动剂对细胞增殖的影响。ERα选择性激动剂PPT,ERβ选择性激动剂DPN及ER非选择性激动剂E2处理组与未经药物处理的对照组相比,细胞增殖情况无差异。说明ER两种受体对原代培养的侵袭性NFPA细胞增殖没有影响。3.利用Transwell小室检测各组细胞的侵袭性,结果显示加入ERα选择性激动剂PPT组及非选择性激动剂E2处理组细胞侵袭性增高(P<0.01),而ERβ选择性激动剂DPN处理组细胞侵袭性受到抑制。4. mRNA水平及蛋白水平检测结果一致表明,ERα受体激活后Slug的表达出现上调,E-cadherin的表达受到抑制;与之相反,ERβ受体激活后Slug的表达收到抑制,E-cadherin表达上调。结论1.临床标本的研究表明ERα在侵袭性NFPA中高表达,对该肿瘤侵袭性起促进作用,同时ERβ表现为对该肿瘤侵袭性起抑制作用。离体实验进一步证实这一效应: ERα受体激活后细胞侵袭性增高,而ERβ激活后细胞侵袭性受到抑制。2.在NFPA组织和原代培养细胞中,E-cadherin表达量的降低使NFPA的侵袭性增高,该蛋白的表达受其抑制子Slug的调控,并与Slug的表达呈负相关。3.临床标本和原代培养细胞的研究表明ER影响NFPA中Slug-E-cadherin通路,其中ERα主要上调Slug的表达,抑制E-cadhein的表达,使肿瘤的侵袭性增高;与之相反,ERβ的作用表现为抑制Slug的表达,从而上调E-cadherin的表达,对肿瘤的侵袭性产生抑制作用。综上所述,ERα与ERβ通过相反的作用调控Slug-E-cadherin通路而影响NFPA的侵袭性,其中ERβ主要表现为抑制作用,ERα表现为促进作用。更多还原

【Abstract】 Pituitary adenomas (PAs) comprise 15 to 20% of primary intracranial tumors. Though they are benign tumors, PAs often invade surrounding structures such as sphenoid sinus and cavernous sinus. Total surgical removal of invasive adenomas remains a challenge for surgeon, for the local invasion of the tumor mass. Due to re-growth of residual tumor, invading PAs are associated with higher recurrence rate and they are considered as an intermediate stage in tumor progression towards carcinomas. Clarification of the factors affecting tumorous invasion is essential for evaluation of prognosis in patients and developing new adjuvant treatment.Estrogen, through ERs (ERαand ERβ), affects cellular proliferation and hormone synthesis in pituitary gland. Recent studies have demonstrated that there is a correlation between ER level and tumor growth. In GH3 cell line, ERαis able to up-regulate the transcription of key growth-promoting genes. Moreover, in female ERβ–/–mice loss of ERβlead to development of large gonadotropin-positive pituitary tumors when the mice are two years of age. As similar as double knockout ERαβ–/– mice, ERα–/– and male ERβ–/–mice do not develop pituitary tumors. These results indicate that an imbalance between ERαand ERβcontributes to pathogenesis and biological behavior of PAs; however, the underlying mechanism is unclear.Disconnection and detachment of cells are crucial for tumor invasion. Loss or down-regulated of E-cadherin may contribute to the development of tumor invasion in a wide variety of human malignancies. In prolactinomas, loss of membranous staining of E-cadherin was frequently associated with tumor size and local invasion. These results imply that E-cadherin level may contribute to invasiveness of PAs. Several other series of studies showed that E-cadherin level was affected by estrogen receptor activation, but no studies have investigated the relationship between E-cadherin and estrogen receptors in pituitary tumors. Slug is a member of the snail family of transcription factors; it repressed the expression of E-cadherin. Both in vitro and in vivo studies have suggested that the expression of E-cadherin is inversely correlated with the expression of slug in tumorigenesis. Although the relationship between slug and E-cadherin are being elucidated in many tumors of epithelial origin, comparatively much is unknown about slug regulation in PAs.The nonfunctional pituitary adenoma (NFPA) is one of the most common PAs. In contrast to other PAs characterized by hormonal excess, NFPAs are characterized by null cells or endocrine-inactive neoplasms of anterior pituitary. In this study, we studied the expressions of ERs in invasive and noninvasive NFPAs tissues using immunohistochemistry and evaluated whether these parameters are associated with tumor invasiveness. Expressions of E-cadherin and Slug, PCNA-labeling index in invasive and noninvasive NFPAs were evaluated as indicators of tumor invasiveness. In addition, the roles of ERαand ERβagonist (PPT and DPN respectively) in the migration and proliferation of primary cultured pituitary adenoma were investigated.The study consists of two parts:Part 1: The expression of ERs in the NFPAsNFPAs tissues were obtained from 41 patients at Xin Qiao Hospital and the patients all had transsphenoidal surgery via a sublabial or transnasal approach. Epidemiological analysis was performed to study the correlation between estrogen level in the serum and invasiveness of NFPAs. The expression of ERαand ERβin the NFPAs were detected by immunohistochemistry, the correlation between invasion and proliferation in NFPAs was evaluated with PCNA and Ki-67 labeling index. In addition, the expression of E-cadherin and its repressor Slug were analyzed to understand the potential mechanism that ERs affect the invasion of NFPAs.Results:1. The occurrence of invasive type was statistically significantly lower among men compared with woman. There is no significant difference in the serum E2 concentrations of patients with invasive NFPAs between females and males, whereas nuclear ERαstaining in the invasive NFPAs was significantly stronger in the female patients than that from male patients. 2. ERαexpression was deteced in 37 of 41 (90%) samples and in all 15 invasive adenomas. For ERβ, nuclear staining in noninvasive adenomas was positive in 25 of 26 (96%) samples, whereas in invasive ones, expression was lost in 9 of 15 (60%) samples. Moreover, compared with noninvasive NFPAs, expression of ERαwas extremely higher in the invasive ones, whereas the expression of ERβwas significantly lower. These results indicate that ERs may play an important role in the regulation of NFPAs invasiveness, in which ERβprobably inhibits this process whereas ERαcontributes to it.3. Strong expression of PCNA was detectable in all 41 samples; however, the PCNA labeling index was not different between invasive NFPAs and noninvasive ones. Similar to expression of PCNA, there is no significant difference in the Ki-67 labeling index between these two groups. Then we infer that proliferation was not always associated with invasion of PAs,nor associated with the expression of ERαand ERβ.4. The levels of E-cadherin and Slug were detected using semi-quantitative RT-PCR. Low expression of E-cadherin was detected in five invasive NFPAs, whereas expression of Slug in the noninvasive NFPAs was high. Slug mRNA was upregulated in the invasive NFPAs compared with noninvasive ones. These differences were confirmed at the protein level by Western blotting and immunohistochemistry. Furthermore, level of Slug was negatively correlated with ERβand positively correlated with ERα; whereas level of E-cadherin was negatively correlated with ERαand positively correlated with ERβ. It seemed that ERs may act on the Slug-E-cadherin pathway to affect invasion of NFPA.Part 2: The effects of ERs agonist on primary culture cells of invasive NFPAsERs are ligand-activated nuclear receptors; their natural ligand is 17β-Estradiol (E2). Two synthetic compounds of PPT and DPN have been identified as special agonists for ERαand ERβrespectively. In this study, endogenous ERs in the primary cultured cells were activated by E2, PPT and DPN administration into culture medium. The proliferation of cells was evaluated by MTT assay and the invasion of the cells was detected by transwell invasion assay. Furthermore, both mRNA and protein levels of E-cadherin and Slug were detected in vehicle and agonist treated groups to analyze the relationship between ERs and E-cadherin or Slug.1. Both ERαand ERβwere expressed in the primary cultured cells derived from invasive NFPAs, of which ERαwas the predominate ER with high expression level. Colocalization of ERαand ERβwas observed with double immunofluorescence, of few double stained cells were detected under microscope.2. There is no significant difference in the cellular proliferation of primary cultured cells derived from invasive NFPAs among agonist treated group and control group. This may infer that ERs activation had no significant effect on NFPA cell proliferation in vitro.3. The invasion of cells was evaluated by transwell invasion assay. We found that cells treated with either ERαagonist PPT or E2 exhibited high invasion, whereas the cells treated with DPN exhibited repressed invasion.4. After treated with ERαagonists PPT or E2, the cells showed upregulated Slug expression and downregulated E-cadherin expression compared with cells treated with vehicle. Conversely, ERβactivation with DPN in the cells induced downregulated Slug expression, and upregulated E-cadherin expression.Conclusions1. In vivo, expression of ERαwas extremely higher in the invasive NFPA compared with noninvasive ones, whereas the expression of ERβwas significantly lower. In vitro, the activation of ERαinduced high invasion of the tumor cells, otherwise, the activation of ERβrepressed the invasiveness. These results indicate that ERs may play an important role in the regulation of NFPAs invasiveness.2. In NFPA tissues, decreased expression of E-cadherin was associated with tumor invasion, and expression of E-cadherin was downregulated by its repressor Slug. Moreover, this was confirmed by in vitro study.3. Both in vivo and vitro, the result showed that ERβactivation induced downregulated Slug expression, and upregulated E-cadherin expression which repressed NFPAs invasiveness, otherwise, ERαactivation act in the opposite direction.In conclusion, the ERαand ERβmay be involved in the invasiveness of NFPA with opposite way, of which ERβinhibits this process whereas ERαcontributes to it. The underlying mechanism is further to be demonstrated to be associated with the Slug-E-cadherin pathway.更多还原